Coexpression of band 3 with protein 4.2 or ankyrin band-3–binding domain in Xenopus oocytes. Band-3 (B3) cRNA was coinjected into oocytes with the indicated concentration of protein 4.2 or Ank-R89 (Ank) cRNA. DNDS-sensitive chloride influx (20 min) was measured 24 hours after injection with cRNA, using groups of 10 to 15 oocytes as described previously.30 Results are shown as means ± SEM for comparisons with the relevant B3 alone sample by Student t test: ***P < .0005, **P < .001, *.002 <P < .005, and + .005 < P < .01. Panel A shows the effects of varying the B3 concentration from 0.5 ng to 5 ng using 0.5 ng to 5 ng protein 4.2. This suggests that the best combination of cRNA to use for observation of the effects of protein 4.2 on B3-specific transport is 0.5 ng B3 cRNA and 1 to 2 ng protein-4.2 cRNA. Panel B demonstrates that neither protein 4.2 nor Ank-R89 have a significant effect on endogenous chloride transport when 0.5 ng to 5 ng of cRNA is injected into oocytes. In comparison, the enhancement effect can be clearly observed when 0.5 ng of band 3 is co-injected with 5 ng protein 4.2. Panel C shows a comparison of the effects of protein 4.2 and Ank-R89 at 0.5 to 5 ng with B3 cRNA concentration held constant at 0.5 ng. Under these conditions, protein 4.2 enhanced B3-specific chloride, whereas Ank-R89 did not increase B3-specific chloride transport. This suggests that the protein-4.2 enhancement effect was due specifically to 4.2 binding to B3 and not simply a response to protein binding at the N-terminus of band 3.
